The tick borne flaviviruses (TBFV) includes Tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Powassan virus and Langat virus (LGTV). The TBFV are listed among the NIAID category B and C lists of priority for research into pathogenesis, treatment and vaccine development. As their name suggests, these viruses are transmitted by ticks, and following infection of humans, cause encephalitis, meningitis or hemorrhagic fevers resulting in approximately 13,000 hospitalizations annually with mortality rates as high as 40%. [unreadable] [unreadable] The TBFV belong to the Family Flaviviridae, genus Flavivirus, which comprise some of the most medically significant emerging and re-emerging pathogens. Other members include the mosquito-borne West Nile virus (WNV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLE), dengue virus (DEN) and yellow fever virus (YFV). Hence, research into the pathogenesis of TBFV will reveal insight into the biology of this globally important group of viruses.[unreadable] [unreadable] The research in our laboratory aims to identify and understand interactions between the TBFV and their hosts (both the tick and the mammal) critical to virus replication and pathogenesis. We have been studying LGTV which is a naturally attenuated member of the TBFV that shares approximately 80% identity with TBEV at the amino acid level. This makes LGTV an excellent model to gain insight into the TBFV, with the aim of identifying specific therapeutic targets. In particular, our laboratory has a number of ongoing projects outlined below to study interactions between the TBFV and their hosts, with an emphasis on host innate immunity.[unreadable] [unreadable] 1. Interactions between TBFV and host innate responses to infection.[unreadable] Following the bite from an infected tick, dendritic cells resident in the skin of the mammalian host are thought to be among the first cell types infected by TBFV. These cells have important roles in innate immunity through the production of interferon (IFN) as well as orchestrating adaptive immunity. The early interactions between these cells and TBFV are likely to have a major influence on the outcome of infection. We have initiated two projects in the lab to investigate 1) effects of TBFV infection on primary dendritic cells and 2) ability of TBFV to modulate the signal transduction pathways involved in detecting virus infection and producing IFN. These projects are in their infancy, although we expect that this work will yield insight into immune evasion mechanisms utilized by the flaviviruses.[unreadable] [unreadable] Type I (IFN and IFN) and type II (IFN) IFNs are crucial elements of the innate immune response to flavivirus infection, restricting virus replication, dissemination and lethality in mouse models. Type I IFN treatment of humans is a leading therapeutic candidate for flavivirus infection. However, such treatment often fails. We have shown that LGTV interferes with IFN signaling by directly inhibiting Janus kinase-signal transducer and activator of transcription (JAK-STAT) signal transduction. In this manner, LGTV is similar to the mosquito-borne flaviviruses WNV, JEV, DEN and YFV, which can also inhibit IFN-mediated JAK-STAT signaling. Our laboratory demonstrated that LGTV suppresses JAK-STAT signaling in response to both type I and type II IFN by inhibiting the phosphorylation of the JAKs. Examination of the ability of each individual nonstructural (NS) protein to prevent JAK-STAT signaling revealed that LGTV NS5 was primarily responsible for inhibiting signal transduction, via associating with IFN receptor complexes. [unreadable] [unreadable] The finding that NS5 functions as an interferon antagonist was unexpected because NS5 has a crucial role in viral RNA replication via its two enzymatic domains, the RNA-dependent RNA polymerase (RdRP) and the methyltransferase. By expressing a series of N- and C-terminal truncation mutants, we determined that the minimal sequence of NS5 required for its IFN-inhibitory function mapped to amino acids 355-735. This region overlaps entirely with the RdRP. By performing functional assays with approximately 350 mutagenized NS5 constructs, we identified a unique functional site on the RdRP responsible for inhibition of JAK-STAT signaling. These are remarkable findings because they indicate at least three functions for the TBFV NS5 V a methyltransferase, the RdRP and the JAK-STAT inhibitory function, with the domains responsible for the latter two overlapping. [unreadable] [unreadable] In addition to the specific studies on LGTV NS5, we have investigated the relative IFN antagonist ability of NS5 and another nonstructural protein NS4B (implicated in IFN antagonism for DEN) from different flaviviruses including WNV and JEV. These studies have identified NS5 as a major IFN antagonist for WNV in addition to the already known role of NS5 in IFN-evasion by JEV. Some of the individual residues required for LGTV NS5 function as an antagonist were demonstrated to be important for function of WNV NS5. Mutations at these residues are now being incorporated by one of our collaborators into live-attenuated vaccines against WNV infection. Taken together, this work will provide further insight into the importance of IFN-evasion in flavivirus pathogenesis as well as contribute to the next generation of live-attenuated vaccine design.[unreadable] [unreadable] 2. Study of virus interactions with the invertebrate host. [unreadable] In addition to working with mouse models of infection, we have developed a novel method of infecting tick larvae with LGTV by immersion. This extremely versatile method permits synchronous infection of large numbers of ticks with a defined virus inoculum, and without a requirement for that virus to establish an infection and viremia in mice. This method not only enables study of virus replication within the initial infected tick larvae and nymphs, but also results in efficient trans-stadial transmission as well as horizontal transmission to C57BL/6 mice. Hence, the immersion method of infection is a powerful tool to study viral and host determinants for pathogenesis in both ticks and in the mammalian host. We are currently utilizing the immersion method of infection to examine global transcriptional responses of the tick, Ixodes scapularis, to infection with LGTV using spotted microarrays. The aim of this work is to identify tick host proteins important for the replication or transmission of TBFV. These proteins can be used for the development of novel anti-tick vaccines that prevent virus transmission to the immunized mammalian host. [unreadable] [unreadable] In contrast to virus infection in laboratory mouse models and in humans, infection of TBFV in tick vectors is persistent without obvious cytopathic effect. We are interested in virus determinants that confer a selective replication advantage in either tick or mammalian systems. To examine this question, we derived a number of LGTV variants by repeatedly passaging the virus in tick or mammalian cell culture, followed by sequencing of the virus genome. We anticipated that virus adaptation to replication in these two cells types would be associated with sequence changes that either alter efficiency of virus replication in other cell types or perhaps alter virulence following inoculation of C57Bl/6 mice. To summarize this work, we identified two clusters of coding changes in the LGTV genome associated with host adaptation. The first was in the envelope (E) protein while the second was in the region encompassing NS3, NS4A and NS4B. We plan to use the two animal models of infection, both tick and mouse, in combination with reverse genetics to determine the relative contribution of these genetic changes to virus transmission and virulence.